Field of Invention
This invention relates to artificially created tissues and methods of making the same, specifically, the invention relates to tissues with improved mass transport capabilities.
Description of Related Art
The shortage of organs for organ transplantation is a serious medical issue. The aging population continues to grow due to several factors such as, for example, the aging of the baby boomers population, improved lifesaving medical techniques and drugs, and a rise of average life expectancy. Tissue engineering is one of the avenues scientists explore to create various artificial organs. One of the major shortcomings of currently available artificial organs is inability to provide sufficient flow of nutrients, transport cells, and a removal of waste to support the growths of cells and development of tissues. Some tissue scaffolds are seeded with cells and implanted in vivo. They rely upon the infiltration and development of natural blood vessels into the scaffold to keep the cells alive. However, these processes may take too long and result in the death of cells, especially those cells located within the interior of the scaffold, wherein cells are starved for nutrients due to the limitations of diffusion.
Prior attempts to improve the flow include creating bioreactors to develop the cells within the tissue scaffold in vitro first, and implanting the tissue construct at a later stage of development.
Many different types of bioreactors have been designed to improve mass transport [1]. Some of the basic types of bioreactors are spinner flasks, various types of rotating cell culture systems (RCCS), and perfusion systems [2]. Cells with higher metabolisms have different requirements from cells with less of a need for mass transport. For example, a rotational, oxygen-permeable bioreactor system (ROBS) was used to culture osteoblasts [3] and chondrocytes [4]. Given the avascular nature of cartilage, such approach would not work for cells with greater metabolic requirements.
The general problem with many of known scaffolds is that the tissue constructs are still very small in size, i.e., only a few millimeters in diameter, and even less in depth for cells having higher metabolic requirements. Larger tissue constructs could be formed, provided that they could be vascularized quickly enough to prevent necrosis in tissue's interior. The creation of a circulatory system is vital for successful in vitro organogenesis.
Further, bioreactors are unnatural environments that do not reflect the conditions of living systems. Cells may be exposed to turbulent flows, which are not conductive for proper cellular development. Numerous inventors have tried to control the environmental conditions to improve cell culture conditions, see for example, U.S. Pat. No. 5,523,228 to Ingram, et al. and U.S. Pat. No. 6,001,643 to Spaulding.
Inventors have tried to imitate nature to help create a better environment for cell culture. Artificial capillaries have been described by Knazek, et al. [5] and Knazek et al., in U.S. Pat. No. 4,200,689 using hollow polymeric fibers that were permeable to gases. However, the problems with such structures include biocompatibility, implantation into a living organism, and limits to the space available for cell growth and expansion. Others have also explored hollow fiber systems and bioreactors (see, for example, U.S. Pat. No. 3,997,396 by Delente, U.S. Pat. No. 5,081,035 by Halberstadt et al., U.S. Pat. No. 5,549,674 by Humes, et al., and U.S. Pat. No. 6,001,585).
Pulsating flow systems have been created (U.S. Pat. No. 6,632,651 by Nevo, et al.) for growing cells in tissue scaffolds to mimic in vivo conditions. However, the circulatory mechanisms were external to the scaffold. There was no internal circulatory system built within the scaffold itself. Living systems have internal circulatory systems. Current tissue scaffolds do not provide for internal circulatory systems filled with materials for improving circulation.
Most tissue scaffolds rely upon diffusion for supplying cells with nutrition. Diffusion is highly limited. For static culture, the depth of the medium should only be 2-5 mm for adequate diffusion of oxygen to the cells [6]. In normal tissue, cells are generally within 200 micrometers at most from their blood supply.
With the current state of technology for tissue scaffolds, methods for improving diffusion generally do not go beyond creating simple channels and pores (see U.S. Pat. No. 6,534,084 to Vykarnam, U.S. Pat. No. 6,423,252 to Chun, et al.) or improving pore structure (see U.S. Pat. No. 6,103,255 to Levene et al., U.S. Pat. No. 6,537,567 to Niklason et al. describing various possibilities for a tissue engineered construct). However, these methods adhere to the traditional paradigm of transport through lumens, tubes, and fibrous meshes. U.S. Pat. No. 6,455,311 by Vacanti describes etching or creating channels and lumens on various substrates and in three-dimensions using various manufacturing techniques. Again, the mindset is still the same. Levene, et al. U.S. Pat. No. 6,337,198 describes their invention of a porous scaffold with an interconnected system of open pores to promote diffusion.
The conventional paradigm is based on using macropores, channels and microchannels as the primary means of diffusion. The problem with these scaffold designs is that channels can become occluded as cells grow and lay down extracellular matrix, thus choking off the flow of nutrients resulting in cellular necrosis occurring within the interior of the scaffold. Currently, it is very difficult to keep a large tissue construct adequately supplied with enough oxygen and nutrients, and to remove cell waste products from their local environment.
Hydrogels, such as alginates, have been used as cell scaffold materials. In the case of alginates, poor cell adhesion has been looked upon as a negative. Researchers have done experiments with chemical modification of alginates, such as adding RGD peptides or doing other chemical modifications to improve cell adhesion (Mooney, et al. in U.S. Pat. No. 6,642,363).
Hubbell, et al. in U.S. Pat. No. 5,330,911 describe the use of RGD, YIGSR, or REDV moieties to improve cell adhesion to surfaces, as well as utilizing PEO to prevent cell adhesion and clotting. Usala in U.S. Pat. No. 6,713,079 uses hydrogel matrix, such as collagen, to promote cell growth, infiltration, and vascularization to improve wound healing.
Hydrogels have been used for cell-encapsulation an example (see, for example, U.S. Pat. No. 5,976,780 to Shah). Hubbell in U.S. Pat. No. 6,129,761 describes the use of hydrogel-cell compositions in an injectable form. Griffith-Cima et al. in U.S. Pat. No. 6,730,298 use hydrogels as a method for cell delivery via encapsulation, and also shaping the injected gel using a mold. Others follow a similar pattern of using hydrogels for cell proliferation, encapsulation, and injection such as Usala, et al. in U.S. Pat. No. 6,730,315. Vacanti, et al. in U.S. Pat. No. 6,027,744 and Vacanti, et al. in U.S. Pat. No. 6,171,610 also use hydrogels as a scaffold material with cells embedded within the hydrogel. The diffusive properties of the hydrogel allow mass transport to occur to and from the cell, thereby keeping the cell alive. However, this also follows the basic idea of cell encapsulation, and cells are suspended within the hydrogel.
They essentially follow the conventional paradigm of using hydrogels for encapsulation and scaffolding for cells. The thought of using the hydrogel as an acellular material designed to function other than as a support or encapsulating matrix is not considered. Mahmood et al. in U.S. Pat. No. 6,692,761 utilize the basic property of hydrogels in allowing diffusion of materials and nutrients. These diffusion properties of hydrogels have been utilized for drug delivery. Dextran hydrogels have been used for drug delivery (see U.S. Pat. No. 6,525,145 to Gevaert, et al.). Antanavich, et al. in U.S. Pat. No. 6,372,244 designed thin sheets of cellular implants to promote diffusion. Their invention was similar to cell encapsulation by alginate. Lyles et al. in U.S. Pat. No. 6,340,360 detailed an implant that used a biodegradable matrix to deliver drugs and biological factors via diffusion. Shapiro, et al. in U.S. Pat. No. 6,334,968 detailed their invention of an alginate sponge for use as a cell matrix, cell scaffold, and for delivery of therapeutic agents.
However, none of these inventions recognized the possibility of creating an artificial transport system utilizing the diffusion properties of hydrogels.
Naughton et al. in U.S. Pat. No. 6,008,049 devised a bioreactor that uses diffusion gradients to simulate a more natural method of supplying nutrients and removing wastes. Cells are seeded onto a mesh, but the mesh merely acts as a scaffolding and support structure, and is not actively involved in transport functions.
Mizuno et al. in U.S. Pat. No. 6,949,252 utilized the capillary effect of a collagen sponge in order to seed the matrix with cells; however, the capillary effect was not designed to provide for a method of mass transport. The capillary effect was to help promote cell seeding.
Takezawa and Takezawa, et al. utilized readily available fibrous meshes to function as a capillary-like network for mass transport. They cultured fibroblasts on a cotton gauze mesh coated with collagen. They used its absorptive capabilities to help circulate fluid throughout the culture by connecting it to a peristaltic pump [7, 8]. In addition, they utilized the roots of a rice plant as a novel scaffold for growing fibroblasts [8]. Takezawa, et al. in U.S. Pat. No 5,736,399 described the use of natural or synthetic threads or meshes to act as a means of improving diffusion within a three-dimensional cell culture. A device could be physically connected to the network to improve transport of culture media to the cells. This method has a disadvantage in that it would be difficult to integrate the diffusive mesh into a tissue scaffold. Also, gels were used as a coating for the mesh.
Layered manufacturing techniques have been applied to the field of biology. This has resulted in much research being conducted within the field of computer-aided tissue engineering (CATE).
Reischmann and Weiss, et al. have described a method for building bone tissue scaffolds using laminated sheets of material and stacking them together [9, 10]. Yan and Xiong et al. have disclosed the concept of using layered manufacturing methods and multi-nozzle deposition extrusion and jetting processes [11, 12]. R. Landers et al. have also devised a SFF method using a syringe-based system to dispense liquids, which is suited for working with biological materials such as cells and hydrogels [13, 14, 15]. Calvert et al. have devised a syringe-based system for the extrusion of hybrid polymer materials embedded with glass using layered SFF manufacturing [16]. Vozzi et al. have devised a microsyringe deposition system [17, 18]. Ang et al. created a single-nozzle, automated extrusion system that can utilize basic STL files [19]. U.S. Pat. No. 6,139,574 (Vacanti et al. Oct. 31, 2000) discloses vascularized tissue regeneration matrices formed by solid free form fabrication techniques. U.S. Pat. No. 6,143,293 (Weiss, et al. Nov. 7, 2000) discloses assembled scaffolds for three dimensional cell culturing and tissue generation. U.S. Pat. No. 6,027,744 and U.S. Pat. No. 6,171,610 (Vacanti, et al. Feb. 22,2000 and Vacanti, et al. Jan. 9,2001) describe guided development and support of hydrogel-cell compositions. U.S. Pat. No. 6,176,874 (Vacanti, et al. Jan. 23, 2001) discloses vascularized tissue regeneration matrices formed by solid free form fabrication techniques. U.S. Pat. No. 6,454,811 (Sherwood, et al. Sep. 24, 2002) discloses composites for tissue regeneration and methods of manufacture thereof. This method primarily focuses on 3DP for tissue engineering. U.S. Pat. No. 6,547,994 (Monkhouse, et al. Apr. 15, 2003) describes a process for rapid prototyping and manufacturing of primarily drug delivery systems with multiple gradients, mostly involving the 3DP technique. U.S. Pat. No. 6,623,687 (Gervasi, et al. Sep. 23,2003) describes a process for making three-dimensional objects by constructing an interlaced lattice construct using SFF to create a functional gradient material.
Tissue scaffolds are limited by a lack of diffusion and circulation. The current state of technology generally relies upon interconnected channels and pores. Attempts to improve circulation are done externally using bioreactors to improve the flow of culture medium around the scaffold. Passive transport, such as a diffusion process, has an advantage in that it does not require the presence of a mechanical apparatus associated with bioreactors and perfusion pumps. Artificial methods for creating internal, diffusive meshes have been devised; however, their integration into a tissue scaffolds is too complex and does not provide reliable and consistent results. However, the presently known scaffolds are still diffusion limited internally and large scaffolds can have necrotic cores.
Thus, despite the foregoing developments, there is still a need in the art for artificial tissues having an improved internal transportation network.
All references cited herein are incorporated herein by reference in their entireties.